In type 2 diabetes, impaired insulin signaling leads to reduced GLUT4 trafficking and thus decreased glucose uptake in fat and muscle. In addition, defects in intracellular GLUT4 targeting, apart from insulin signaling defects, may also contribute to diabetes pathophysiology. In cultured 3T3-L1 adipocytes, GLUT4 resides in several compartments consistent with the itinerary of a recycled membrane protein. However, unlike other recycled proteins, GLUT4 is efficiently sequestered away from the plasma membrane in cells not stimulated with insulin, and instead accumulates in a population of small, specialized vesicles termed GLUT4 Storage Vesicles (GSVs). TUG is highly expressed in adipocytes and skeletal muscle and acts to tether GLUT4 intracellularly in the absence of insulin, excluding the glucose transporter from the plasma membrane in 3T3-L1 adipocytes. Upon insulin stimulation, the interaction between TUG and GLUT4 is disrupted. GLUT4 "untethers" from its specialized internal membrane compartment, and initiates its translocated to the plasma membrane to promote glucose uptake, it is unknown how and when TUG becomes associated with GSVs. One hypothesis promotes that TUG is recruited to these vesicles during their biogenesis. Indeed, preliminary data presented in this proposal demonstrates that TUG interacts with an atypical clathrin adaptor (GGA2) previously shown to be essential for GSV formation in 3T3-L1 adipocytes. It is hypothesized that by binding to GLUT4 and the clathrin adaptor, TUG may help to specify GSV cargo. In addition, by participating in the budding process, TUG is primed to function as a retention factor for newly formed GSVs. As part of a larger effort to understand how GGA2 and TUG function together to control GLUT4 trafficking, three Specific Aims will be performed. First, the GGA2-TUG interaction will be characterized in detail, using both recombinant proteins and immunofluorescence microscopy of 3T3-L1 adipocytes. Second, disruption of TUG in these 3T3-L1 cells will be used to study effects on GGA2-dependent GSV budding. Third, a direct role for TUG in GSV formation will be examined by reconstituting GSV budding in a controlled in vitro environment. The incidence of type 2 diabetes in America is reaching epidemic proportions while the cellular pathogenesis of this disease remains unresolved. It is anticipated that the results of this work will have importance for understanding how GLUT4 glucose transporters are properly sorted from internal membranes into the insulin responsive compartment.